Metabolomics

Metabolomics and lipidomics data derived from serum and brain samples

Metabolomics data is generated by the research team lead by Dr. Rima Kaddurah-Daouk. The data is available for a subset of ROS and MAP participants. MAP lipidomics data generated by Phenomenome Discoveries, Inc.

Subcategories

Bile acid

Quantification of bile acid concentration was performed at the University of Hawaii cancer center. The bile acid-free matrix (BAFM) was used to prepare bile acid calibrators. Extracts of brain tissue along with bile acid reference standards were subjected to instrumental analysis. A Waters ACQUITY ultra performance LC system coupled with a Waters XEVO TQ-S mass spectrometer was used for all analyses. Chromatographic separations were performed with an ACQUITY BEH C18 column.

Sample analysis:

UPLC-MS raw data obtained with negative mode were analyzed using TargetLynx™ applications manager to obtain calibration equations and the quantitative concentration of each bile acid.

Brain metabolomics

Brain metabolomics data is available for 111 ROS and MAP participants.

Weighed brain tissue samples were provided frozen. Each sample was diluted with 100 μL 1:1 methanol:water. Samples were then homogenized using 3 10 second pulses in the Precellys Evolution between which samples were chilled in an ice bath. After the first 3 cycles 250 μL 3:1 methanol:chloroform were added to each sample followed by 3 10 second pulses between which samples were again chilled in an ice bath. The samples were then centrifuged at 15000 rpm for 10 minutes in a refrigerated (4C) centrifuge. The homogenized samples were stored at -80C until the day of extraction with the p180 kits. In order to eliminate some potential extraction inefficiencies observed with the glycerophospholipids, the homogenized samples remaining from the original analysis in Project 4693 were normalized to a concentration of 0.1 mg/μL. This was accomplished by adding the appropriate volume of combined extraction solvents (1:2.5 v/v (1:1 MeOH:Water, 3:1 MeOH:Chloroform). After solvent addition samples were homogenized using three 10-second pulses in the Precellys Evolution between which samples were chilled on ice. On the day of sample extraction, the samples were thawed and centrifuged at 15000 rpm for 10 minutes, and then stored on ice until addition to the p180 kit plates.

Samples were prepared using the AbsoluteIDQ p180 kit (Biocrates Innsbruck, Austria) in strict accordance with their detailed protocol. After the addition of 10 μL of the supplied internal standard solution to each well of the 96-well extraction plate, 10 μL of each blank, calibration standard, QC sample, and study sample were added to the appropriate wells. The plate was then dried under a gentle stream of nitrogen for 10 minutes. An additional 10 μL of each SPQC and brain tissue homogenate sample were added to the appropriate wells followed by an additional 20 minutes of drying. The samples were derivatized with phenyl isothiocyanate then eluted with 5mM ammonium acetate in methanol. Samples were diluted with either 1:1 methanol:water for the UPLC analysis (4:1) or running solvent (a proprietary mixture provided by Biocrates) for flow injection analysis (20:1).

Sample analysis:

Sample analysis of amino acids and biogenic amines was performed using UPLC (ultra-high pressure liquid chromatography). Acylcarnitines, sphingolipids, and glycerophospholipids were analyzed by FIA (flow injection analysis). Using electrospray ionization in positive mode, samples for both UPLC and flow injection analysis were introduced directly into a Xevo TQ-S triple quadrupole mass spectrometer (Waters) operating in the Multiple Reaction Monitoring (MRM) mode. MRM transitions (compound-specific precursor to product ion transitions) for each analyte and internal standard were collected over the appropriate retention time. The UPLC-MS/MS data were imported into Waters application TargetLynx™ for peak integration, calibration, and concentration calculations. The UPLC-MS/MS data from TargetLynx™ and FIA-MS/MS data were analyzed using Biocrates MetIDQ™ software.

Serum metabolomics

Serum metabolomics data is available for a subset of ROS and MAP participants. 6 of the 603 blood samples were less than 10μL. The remaining 597 samples came from 560 ROS/MAP participants (repeated measures were available in 31 participants). A total of 92 participants had both brain and blood metabolomics data.

Samples were prepared using the AbsoluteIDQ p180 kit (Biocrates Innsbruck, Austria) in strict accordance with their detailed protocol. After the addition of 10 μL of the supplied internal standard solution to each well of the 96-well extraction plate, 10 μL of each study sample were added to the appropriate wells. The plate was then dried under a gentle stream of nitrogen. The samples were derivatized with phenyl isothiocyanate then eluted with 5mM ammonium acetate in methanol. Samples were diluted with either 1:1 methanol:water for the UPLC analysis (4:1) or running solvent (a proprietary mixture provided by Biocrates) for flow injection analysis (20:1).

Sample analysis:

Sample analysis of amino acids and biogenic amines was performed using UPLC (ultra-high pressure liquid chromatography). Acylcarnitines, sphingolipids, and glycerophospholipids were analyzed by FIA (flow injection analysis). Using electrospray ionization in positive mode, samples for both UPLC and flow injection analysis were introduced directly into a Xevo TQ-S triple quadrupole mass spectrometer (Waters) operating in the Multiple Reaction Monitoring (MRM) mode. MRM transitions (compound-specific precursor to product ion transitions) for each analyte and internal standard were collected over the appropriate retention time. The UPLC-MS/MS data were imported into Waters application TargetLynx™ for peak integration, calibration, and concentration calculations. The UPLC-MS/MS data from TargetLynx™ and FIA-MS/MS data were analyzed using Biocrates MetIDQ™ software.

Brain lipidomics

Lipid levels were measured from post-mortem samples of frontal cortex, temporal cortex, and cerebellum of a subset of MAP participants. Biospecimens were transferred to Phenomenome Discoveries, Inc. in 2008 where lipid levels were measured by liquid chromatography-mass spectrometry.

Serum lipidomics

Lipid levels were measured from serum samples of a subset of MAP participants. Biospecimens were transferred to Phenomenome Discoveries, Inc. in 2008 where lipid levels were measured by liquid chromatography-mass spectrometry.